Delayed-type hypersensitivity (DTH) responses are a model of CD4+ T cell recruitment into the tissues in immune resistance and in allergic and autoimmune diseases. The presence of sensitized CD4+, DTH effector T cells in the circulation is not sufficient for recruitment into a tissue site challenged with antigen (Ag). Another Ag-specific cell called DTH- initiating cells is required. These cells elaborate Ag-specific factors that are analogous to IgE antibody and mediate DTH-initiation by leading to local release of the vasoactive amine serotonin. Using a panel of monoclonal antibodies to deplete DTH-initiating activity, an unusual phenotype for an Ag-specific cell was found: Thy-1+, CD5+, CD4-, CD8-, CD3epsilon-, sIg-, B220+, I1-2R-, and IL-3R+. Furthermore, DTH-initiating cells were induced in athymic nude mice but not in SCID mice, leading to the conclusion that DTH-initiating cells were primitive, relatively thymic independent, Ag-specific cells that employed rearranging genes to encode the Ag-specific factors that mediate DTH-initiation. Nude mice were used to immunize and boost DTH-initiating cells in the absence of contaminating CD4+ DTH-effector T cells, or CD8+ suppressor T cells that down-regulate DTH-initiating cells, and in vitro lines and clones of DTH-initiating cells were generated. the phenotype of the DTH- clones determined by FACS and molecular analysis confirmed that DTH-initiating cells are primitive Ag- specific cells of a unique mixed phenotype: for T-like markers: Thy-1+, CD5+, CD4-, CD8-, surface and mRNA CD3-, surface and mRNA alphabeta TCR- and delta TCR mRNA+; for B-like markers: surface and mRNA Ig-, B220+, CD23-; and for Mphi markers FcgammaR+, Mac1+, Class II+. The current specific aims are to: 1) Develop DTH-initiating clones of another Ag specificity to confirm Ag-specificity of DTH-initiation at the clonal level. 2) To clone, sequence, and express the product of the TCR- delta hybridizing gene that is transcribed in the DTH-initiating clones. 3) To clone the gene(s) that encode the DTH-initiating factors. An unamplified cDNA library will be constructed from the DTH-initiating clone in lambdalZAP and will be screened by several means to attempt to isolate cDNA(s) encoding the Ag specific DTH-initiating factor. Probing will be attempted with a polyclonal antibody for DTH-initiating factors, and also with a ligand probe consisting of a conjugate of the relevant hapten coupled to albumin to which is also linked alkaline phosphatase. Attempts will continue to biochemically purify the DTH-initiating factor to obtain amino acid sequence information to construct an oligonucleotide probe. Finally, hamster monoclonal antibodies will be developed to DTH-initiating factor to provide monospecific reagents to screen the library. It is thus hoped that knowledge will be advanced of the important in vivo phenomena of DTH-initiation to the level of molecular cloning of the biologically relevant molecule.